The Zbtb family of proteins consists of more than 40 members in mice. They are highly conserved transcriptional repressors that contain a BTB (POZ) protein-binding domain and a Kruppel-type Zinc Finger (ZF) DNA-binding domain. Although the function of most Zbtb members is still unknown, many Zbtb proteins have essential functions during development, and some were identified as controlling unique differentiation pathways in lymphocytes. For example, Th-POK is a master regulator of CD4 versus CD8 differentiation of T cells, Lrf (Pokemon), was shown to direct B cell versus T cell differentiation, Bcl6 is necessary for the germinal center reaction in B cells and directs the differentiation of CD4 T cells towards T follicular helper (Tfh) cells, and we and others have identified that increased expression of PLZF is necessary for the acquisition of effector functions during the differentiation of Natural Killer T (iNKT) cells, a subtype of T lymphocytes with innate and cytotoxic functions. The potential ability of different Zbtb proteins to interact with each other and to regulate transcription on multiple targets sites is expected to generate a transcriptional regulatory network of which very little is known, mostly because of the still uncharacterized immune function of most Zbtb proteins. The general goal of this project is to elucidate the contribution of specific Zbtb family members to lymphocyte differentiation and function. To this aim, we are focusing on two transcription factors that belong to this family. a) PLZF function and innate-like differentiation of lymphocytes. iNKT cells are a subtype of lymphocytes with unique characteristics, due to their fast activation response, their cytokine-secreting capabilities and their cytotoxic functions, they can shape immune responses in several pathological conditions such as in infection, autoimmunity and cancer. Previous work from us and others identified a correlation between PLZF expression and increased IL-4 production in lymphocytes, however the mechanisms by which PLZF exert these effects are currently unknown. Our data using PLZF-reporter mice indicate that PLZF expression is not only present in iNKT cells but also in other immune cell types. We have also found that PLZF expression is transiently increased during the earlier stages of T-cell development. We have also found that mice lacking PLZF present a reduced total thymic cellularity, suggesting that PLZF expression may play a role during the development of conventional T-cells as well as iNKT cells. We are evaluating the relevance of PLZF expression to the development of immune cells in vivo using several animal systems as for example reconstitution of the immune system in bone marrow chimeras and lineage fate-mapping to identify cells that have expressed PLZF during their differentiation. Our results indicate that some T as well as B lymphocytes are derived from PLZF-expressing progenitor cells. As high PLZF expression was observed in Fetal but not adult hematopoietic progenitors, these results open the possibility that the daughters of PLZF-positive progenitors may possess different phenotypic characteristics than those of PLZF-negative cells. We are in the process of evaluating the phenotype and function of these cells as well as better identifying the role of PLZF-expression at earlier stages of development. b) Another focus of the laboratory is the role of Zbtb1 in T-cell development. It was recently shown that Zbtb1 expression is necessary for the development of lymphoid but not myeloid T-cells. Mice mutant or deficient for Zbtb1 lacked T-cells and have a partial developmental defect in B and NK cell lineages. The deficiency of Zbtb1 led to a T-cell developmental block similar to that of mice deficient in IL-2Rgc cytokines. To study the role of Zbtb1 expression in T-cell development we are identifying its DNA targets. To identify its cellular function we are overexpressing Zbtb1 in immune cell and also analyzing the developmental consequences of Zbtb1-deficiency and overexpression using Zbtb1 mutant mice.